Heat-shock proteins (HSPs) from both the HSP70 and HSP90 families have been shown to functions as tumor-specific antigens, eliciting T cell responses. The antigenicity of HSPs arises from bound peptides and not from their own sequences. Thus, the hypothesis that was developed is that HSPs escort bound antigenic peptides to the ER, where the peptides are transferred to immature MHC class I molecules for presentation to cytotoxic T cells. This hypothesis can account for the ability of purified GRP94, an HSP90 homologue, to enhance the presentation of antigenic peptides to specific T cell clones, when given to macrophages together with these peptides. The potential ramifications of this hypothesis are such that a thorough examination of the biology underlying this exciting phenomenon is needed. Once the mechanistic basis is understood, it may be possible to use this pathway to treat tumors. Accordingly, we propose to test several aspects of this 're-presentation' of peptides by HSPs. First, we propose to determine whether BiP and GRP94, two HSPs localized in the R, can bind known MHC class I antigenic peptides. In particular, we will test in vitro binding of the well- characterized tumor-specific peptides from promiscuous each HSP is with respect to antigenic peptides. Second, we will use microscopy and biochemistry macrophages and dendritic cells. Third, using a non-releasing mutant BiP, we will determine whether peptide is actively transferred from the HSP to MHC class I, and by using antigen presenting cells from TAP- deficient mice, whether peptide needs be transported into the ER to be presented in this pathway. Finally, two approaches will be explored as means of utilizing the peptide re-presentation pathway to augment CTL responses against tumors. First, GRP94 will be genetically engineered to act as a cell surface receptor on antigen presenting cells and test whether such expression increases the presentation capacity of the cells. Second, peptides shown to be good binders to GRP94 and/or BiP will be used in fusions with tumor antigens to enhance specific CTL responses. When integrated with the other projects in the Program, this work will seek to characterize another way to potentiate immune responses against otherwise weak antigens.